The improvement of plant growth by the application of organic fertilizers has been known and carried out for centuries (H. Marschner, "Mineral Nutrition of Higher Plants," Academic Press: New York pg. 674 (1986). Modern man has developed a complex inorganic fertilizer production system to produce an easy product that growers and farmers can apply to soils or growing crops to improve performance by way of growth enhancement. Plant size, coloration, maturation, and yield may all be improved by the application of fertilizer products. Inorganic fertilizers include such commonly applied chemicals as ammonium nitrate. Organic fertilizers may include animal manures and composted lawn debris, among many other sources.
In most recent years, researchers have sought to improve plant growth through the use of biological products. Insect and disease control agents such as Beauveria bassiana and Trichoderma harizamum have been registered for the control of insect and disease problems and thereby indirectly improve plant growth and performance (Fravel et al., "Formulation of Microorganisms to Control Plant Diseases," Formulation of Microbial Biopesticides, Beneficial Microorganisms, and Nematodes, H. D. Burges, ed. Chapman and Hall: London (1996).
There is some indication of direct plant growth enhancement by way of microbial application or microbial by-products. Nodulating bacteria have been added to seeds of leguminous crops when introduced to a new site (Weaver et al., "Rhizobium," Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties, 2nd ed., American Society of Agronomy: Madison (1982)). These bacteria may improve the nodulation efficiency of the plant and thereby improve the plant's ability to convert free nitrogen into a usable form, a process called nitrogen fixation. Non-leguminous crops do not, as a rule, benefit from such treatment. Added bacteria such as Rhizobium directly parasitize the root hairs, then begin a mutualistic relationship by providing benefit to the plant while receiving protection and sustenance.
Mycorrhizal fungi have also been recognized as necessary microorganisms for optional growth of many crops, especially conifers in nutrient-depleted soils. Mechanisms including biosynthesis of plant hormones (Frankenberger et al., "Biosynthesis of Indole-3-Acetic Acid by the Pine Ectomycorrhizal Fungas Pisolithus tinctorius," Appl. Environ. Microbiol. 53:2908-13 (1987)), increased uptake of minerals (Harley et al., "The Uptake of Phosphate by Excised Mycorrhizal Roots of Beech," New Phytologist 49:388-97 (1950) and Harley et al., "The Uptake of Phosphate by Excised Mycorrhizal Roots of Beech. IV. The Effect of Oxygen Concentration Upon Host and Fungus," New Phytologist 52:124-32 (1953)), and water (A. B. Hatch, "The Physical Basis of Mycotrophy in Pinus," Black Rock Forest Bull. No. 6, 168 pp. (1937)) have been postulated. Mycorrhizal fungi have not achieved the common frequency of use that modulating bacteria have due to variable and inconsistent results with any given mycorrhizal strain and the difficulty of study of the organisms.
Plant growth-promoting rhizobacteria ("PGPR") have been recognized in recent years for improving plant growth and development. Hypothetical mechanisms range from direct influences (e.g., increased nutrient uptake) to indirect mechanisms (e.g., pathogen displacement). Growth enhancement by application of a PGPR generally refers to inoculation with a live bacterium to the root system and achieving improved growth through bacterium-produced hormonal effects, siderophores, or by prevention of disease through antibiotic production, or competition. In all of the above cases, the result is effected through root colonization, sometimes through the application of seed coatings. There is limited information to suggest that some PGPR strains may be direct growth promoters that enhance root elongation under gnotobiotic conditions (Anderson et al., "Responses of Bean to Root Colonization With Pseudomonas putida in a Hydroponic System," Phytopathology 75:992-95 (1985), Lifshitz et al., "Growth Promotion of Canola (rapeseed) Seedlings by a Strain of Pseudomonas putida Under Gnotobiotic Conditions," Can. J. Microbiol. 33:390-95 (1987), Young et al., "PGPR: Is There Relationship Between Plant Growth Regulators and the Stimulation of Plant Growth or Biological Activity?," Promoting Rhizobacteria: Progress and Prospects, Second International Workshop on Plant Growth-promoting Rhizobacteria, pp. 182-86 (1991), Loper et al., "Influence of Bacterial Sources of Indole-3-Acetic Acid on Root Elongation of Sugar Beet," Phytopathology 76:386-89 (1986), and Muller et al., "Hormonal Interactions in the Rhizosphere of Maize (Zea mays L.) and Their Effect on Plant Development," Z. Pflanzenernahrung Bodenkunde 152:247-54 (1989); however, the production of plant growth regulators has been proposed as the mechanism mediating these effects. Many bacteria produce various plant growth regulators in vitro (Atzorn et al., "Production of Gibberellins and Indole-3-Acetic Acid by Rhizobium phaseoli in Relation to Nodulation of Phaseolus vulgaris Roots," Planta 175:532-38 (1988) and M. E. Brown, "Plant Growth Substances Produced by Micro-Organism of Solid and Rhizosphere," J. Appl. Bact. 35:443-51 (1972)) or antibiotics (Gardner et al., "Growth Promotion and Inhibition by Antibiotic-Producing Fluorescent Pseudomonads on Citrus Roots," Plant Soil 77:103-13 (1984)). Siderphore production is another mechanism proposed for some PGPR strains (Ahl et al., "Iron Bound-Siderophores, Cyanic Acid, and Antibiotics Involved in Suppression of Thievaliopsis basicola by a Pseudomonas fluorescens Strain," J. Phytopathol. 116:121-34 (1986), Kloepper et al., "Enhanced Plant Growth by Siderophores Produced by Plant Growth-Promoting Rhizobacteria," Nature 286:885-86 (1980), and Kloepper et al., "Pseudomonas siderophores: A Mechanism Explaining Disease-Suppressive Soils," Curr. Microbiol. 4:317-20 (1980)). The colonization of root surfaces and thus the direct competition with pathogenic bacteria on the surfaces is another mechanism of action (Kloepper et al., "Relationship of in vitro Antibiosis of Plant Growth-Promoting Rhizobacteria to Plant Growth and the Displacement of Root Microflora," Phytopathology 71:1020-24 (1981), Weller, et al., "Increased Growth of Wheat by Seed Treatments With Fluorescent Pseudomonads, and Implications of Pythium Control," Can. J. Microbiol. 8:328-34 (1986), and Suslow et al., "Rhizobacteria of Sugar Beets: Effects of Seed Application and Root Colonization on Yield," Phytopathology 72:199-206 (1982)). Canola (rapeseed) studies have indicated PGPR increased plant growth parameters including yields, seedling emergence and vigor, early-season plant growth (number of leaves and length of main runner), and leaf area (Kloepper et al., "Plant Growth-Promoting Rhizobacteria on Canola (rapeseed)," Plant Disease 72:42-46 (1988)). Studies with potato indicated greater yields when Pseudomonas strains were applied to seed potatoes (Burr et al., "Increased Potato Yields by Treatment of Seed Pieces With Specific Strains of Pseudomonas Fluorescens and P. putida," Phytopathology 68:1377-83 (1978), Kloepper et al., "Effect of Seed Piece Inoculation With Plant Growth-Promoting Rhizobacteria on Populations of Erwinia carotovora on Potato Roots and in Daughter Tubers," Phytopathology 73:217-19 (1983), Geels et al., "Reduction of Yield Depressions in High Frequency Potato Cropping Soil After Seed Tuber Treatments With Antagonistic Fluorescent Pseudomonas spp.," Phytopathol. Z. 108:207-38 (1983), Howie et al., "Rhizobacteria: Influence of Cultivar and Soil Type on Plant Growth and Yield of Potato," Soil Biol. Biochem. 15:127-32 (1983), and Vrany et al., "Growth and Yield of Potato Plants Inoculated With Rhizosphere Bacteria," Folia Microbiol. 29:248-53 (1984)). Yield increase was apparently due to the competitive effects of the PGPR to eliminate pathogenic bacteria on the seed tuber, possibly by antibiosis (Kloepper et al., "Effect of Seed Piece Inoculation With Plant Growth-Promoting Rhizobacteria on Populations of Erwinia carotovora on Potato Roots and in Daughter Tubers," Phytopathology 73:217-19 (1983), Kloepper et al., "Effects of Rhizosphere Colonization by Plant Growth-Promoting Rhizobacteria on Potato Plant Development and Yield," Phytopathology 70:1078-82 (1980), Kloepper et al., "Emergence-Promoting Rhizobacteria: Description and Implications for Agriculture," pp. 155-164, Iron, Siderophores, and Plant Disease, T. R. Swinburne, ed. Plenum, New York (1986), and Kloepper et al., "Relationship of in vitro Antibiosis of Plant Growth-Promoting Rhizobacteria to Plant Growth and the Displacement of Root Microflora," Phytopathology 71:1020-24 (1981)). In several studies, plant emergence was improved using PGPR (Tipping et al., "Development of Emergence-Promoting Rhizobacteria for Supersweet Corn," Phytopathology 76:938-41 (1990) (abstract) and Kloepper et al., "Emergence-Promoting Rhizobacteria: Description and Implications for Agriculture," pp. 155-164, Iron, Siderophores, and Plant Disease, T. R. Swinburne, ed. Plenum, New York (1986)). Numerous other studies indicated improved plant health upon treatment with rhizobacteria, due to biocontrol of plant pathogens (B. Schippers, "Biological Control of Pathogens With Rhizobacteria," Phil. Trans. R. Soc. Lond. B. 318:283-93 (1988), Schroth et al., "Disease-Suppressive Soil and Root-Colonizing Bacteria," Science 216:1376-81 (1982), Stutz et al., "Naturally Occurring Fluorescent Pseudomonads Involved in Suppression of Black Root Rot of Tobacco," Phytopathology 76:181-85 (1986), and D. M. Weller, "Biological Control of Soilborne Plant Pathogens in the Rhizosphere With Bacteria," Annu. Rev. Phytopathol. 26:379-407 (1988)).
Pathogen-induced immunization of a plant has been found to promote growth. Injection of Peronospora tabacina externally to tobacco xylem not only alleviated stunting but also promoted growth and development. Immunized tobacco plants, in both greenhouse and field experiments, were approximately 40% taller, had a 40% increase in dry weight, a 30% increase in fresh weight, and 4-6 more leaves than control plants (Tuzun, S., et al., "The Effect of Stem Injection with Peronospora tabacina and Metalaxyl Treatment on Growth of Tobacco and Protection Against Blue Mould in the Field," Phytopathology, 74:804 (1984). These plants flowered approximately 2-3 weeks earlier than control plants (Tuzun, S., et al., "Movement of a Factor in Tobacco Infected with Peronospora tabacina Adam which Systemically Protects Against Blue Mould," Physiological Plant Pathology, 26:321-30 (1985)).
The present invention is directed to an improvement over prior plant growth enhancement procedures.